VEGF-A (VEGF) signaling is essential for regulation of angiogenesis during development and is important for vascular support of tissues in regenerative medicine. Despite extensive studies, we do not understand how VEGF signaling is spatially and temporally regulated at a level that provides useful information in regenerative reconstructions and for treatment in patients. We will build on a strong track record investigating the role of Flt-1 (VEGFR-1) as an endothelial cell-autonomous negative regulator of VEGF-A signaling, and investigate the novel hypothesis that spatial and temporal regulation of Flt-1 trafficking is essential to proper blood vessel formation during development. Our preliminary data shows that both Flt-1 isoforms, the full-length membrane localized receptor (mFlt) and a secreted isoform produced by alternative splicing (sFlt), have regulated trafficking in EC and in angiogenic sprouts. We find that mFlt is highly stable, and we identify a novel interaction between sFlt and a defined secretory trafficking pathway required for proper sFlt secretion. We will use these unique and timely findings as a starting point to thoroughly investigate how Flt-1 isoforms are spatially organized and temporally regulated in EC during sprouting in vitro and in vivo, how sprout connection is regulated by Flt-1 trafficking, and how Flt-1 trafficking and stability affects blood vessel formation. With innovative approaches and diverse models, this renewal will explore the novel idea that Flt-1 trafficking is regulated in space and time to affect sprouting angiogenesis. This work is highly innovative in its focus on trafficking parameters of endothelial cells in biological 3D contexts and in vivo. The expertise and strong track record of this research group, and their commitment to understanding the role of Flt-1 trafficking in vascular development, provide a comprehensive and integrated approach to important unanswered questions. Significant collaborators and consultants will provide expertise. This work will provide new knowledge and unique targets for more precise spatial and temporal manipulation of VEGF-A signaling in therapeutic and regenerative contexts.